Polygalacturonase
In recent years Lycopersicon esculentum, the cultivated tomato, has become a popular system for studying fruit ripening. Tomato fruit ripening is characterized by a series of coordinated biochemical and physiological changes within the various subcellular compartments of the fruit tissue. These changes collectively contribute to the overall quality of the ripe fruit. The most obvious of the changes are alterations in fruit color, flavor, texture and resistance to certain pathogens.
One biochemical change in ripening fruit is the depolymerization and solubilization of cell wall polyuronides by the ripening-induced cell wall degrading enzyme, polygalacturonase (PG). PG activity increases dramatically during the ripening of many fruits, including tomato, and is the primary enzymic activity responsible for cell wall polyuronide degradation during fruit ripening. Reviewed in Giovannoni, et al, 1991 Ann. Rev. Hortic Sci: 67-103.
PG activity isolated from ripe tomato fruit is due to the presence of three structurally and immunologically-related isoforms of PG. These isoforms are termed PG1, PG2A and PG2B. (Ali, et al. Aust. J. Plant Physiol. 9:171, 1982). The PG2A and PG2B isoforms (45 and 46 kDa, respectively) appear well after the onset of ripening and are each composed of a single catalytic PG polypeptide differing only in degree of glycosylation. Because of the physical and biochemical similarity of PG2A and PG2B, the two isoforms shall be treated herein as a single isoform activity (the PG2 activity).
The PG1 isoform (approximately 100 kDa) is the first isoform to appear, at the onset of ripening, and is a heterodimer composed of the single catalytic PG2 polypeptide (either PG2A or PG2B) tightly associated with an ancillary cell wall glycoprotein, the PG beta-subunit. The formation of PG1 by association of the PG2 polypeptide with the PG beta-subunit protein alters both the biochemical and enzymic properties of the associated catalytic PG2 protein. The isoelectric point and pH optimum of PG1 are both a full unit lower than those of PG2. PG1 is more thermo-stable than PG2. PG1 retains complete activity after heating for 5 minutes at 65.degree. C., a treatment that completely inactivates PG2.
In recent years, cDNA clones for the catalytic PG2 polypeptide have been identified and used to examine in detail the regulation of PG gene expression in wild-type and mutant tomato fruit (DellaPenna, et al, Proc. Natl. Acad. Sci. USA 83:6420 (1986). Analysis of PG2 genomic and cDNA clones has revealed that the catalytic PG polypeptide is encoded by a single gene which is transcriptionally activated at the onset of wild-type fruit ripening (DellaPenna et al, Plant Physiol. 90:1372 (1989). PG2 mRNA is synthesized de novo during the ripening of wild-type fruit and accumulates to high levels, accounting for greater than 1% of the mRNA mass. Ripening-impaired mutants of tomato, which are inhibited in many ripening processes including PG2 expression, have greatly reduced levels of PG2 mRNA. The severe reduction in steady-state PG mRNA levels in the mutant genotypes is due to greatly reduced transcriptional activity of the PG gene (DellaPenna, et al, 1989, supra).
The PG beta-subunit has also been studied. The levels of PG beta-subunit increase approximately 4-fold during fruit ripening (Pressey, R., Eur. J. Biochem. 144:217-221 (1984)) and apparently determine the amount of PG1 produced during tomato ripening. Therefore, as PG beta-subunit levels are depleted (by formation of PG1), the timing of appearance of the PG2 isoform is also controlled.
While it is clear from in vitro studies that PG1 and PG2 differ in their biochemical properties, the physiological significance of the isoforms and the role of the PG beta-subunit protein remains uncertain. From a physiological point of view, it seems likely that a cell wall enzyme like PG might be localized or its activity restricted to specific regions of the cell wall by association with an adhesion or localizing factor, such as the PG beta-subunit protein. Recent results in transgenic systems have also suggested that PG1 and hence the beta-subunit, play an important role in vivo with regard to pectin degradation and solubilization, presumably due to its association with the PG beta-subunit protein.
Pectolytic enzymes, such as PG, may have a role in plant pathogen interactions. Pathogen-derived pectolytic enzymes are thought to be important components of the mechanism by which pathogens penetrate and colonize plant tissues. Preliminary results from recently completed experiments have suggested that PG induction in transgenic mutant fruit increases colonization of the fruit by Alternaria alternata, a common late-season pathogen of wild-type tomato fruit to which mutant fruit are normally resistant. The apparent conferral of pathogen sensitivity to mutant fruit by the specific induction of PG expression suggests that increasing PG activity during fruit ripening may play an important role in altering the susceptibility of the fruit to pathogens.
In addition, antisense inhibition of PG expression in wild-type tomato has been correlated with a decrease in "fieldrot" during later stages of tomato ripening in the field. (Kramer, et al, 1990, Horticultural Biotechnology, pp 347-355, Wiley-Liss Inc.). Although the mechanism of PG associated alterations in pathogen susceptibility is not known, these results strongly suggest a role for PG in postharvest pathogenesis.
Tomato Processing
An important determinant of many processed tomato fruit products, including sauce, paste and catsup, is the viscosity (i.e. thickness) of the final product. One of the primary determinants of high viscosity is the presence of large, unmodified pectin molecules. Pectin is a naturally occurring plant cell wall carbohydrate polymer that is composed primarily of polygalacturonic acid residues. Maintenance of pectin integrity during tomato processing is an extremely important part of the commercial process.
An important factor in loss of pectin integrity (decrease in the polymer size and subsequent loss of viscosity) during commercial processing of tomatoes is enzymatic degradation of pectin by PG. Although some modification of pectins by PG occurs naturally during the ripening process (DellaPenna, et al, Plant Physiology 94:1882-86, (1990)), by far the most dramatic and commercially damaging action of PG on pectins and, hence, viscosity occurs when the tomato fruit is homogenized for processing. The PG enzyme present in the fruit has the potential to act in an uncontrolled fashion in homogenized fruit tissues and can rapidly degrade pectin polymers.
A rapid, high-temperature heat treatment is used in commercial tomato processing to destroy PG enzyme activity and thereby maintain a higher viscosity in the final product. This treatment often comprises a process known as "hot break" and is performed by the rapid heating of the tomato product to near boiling point, to inactivate the PG enzyme as rapidly as possible. The annual cost associated with the input of large amounts of energy to bring millions of tons of tomatoes to the temperature needed to rapidly inactivate PG represents a significant cost to tomato processing industries. Annual tomato production in the U.S. is approximately 6 million metric tons representing approximately 10% of worldwide production (1980 figures).
It follows that any process that would allow less energy to be used to inactivate PG in tomato products would result in substantial savings to the industry. A process that would decrease the thermal stability of the PG isoforms would therefore decrease the minimum temperature needed to heat-inactivate PG during processing. All commercially useful, non-genetically engineered tomato varieties currently on the market contain both PG1 and PG2 isoforms. Generally, 10-30% of total PG activity is PG1 in a ripe fruit. One way to decrease the thermal stability of the PG isoforms would be to inactivate or lessen the amount of PG1, the more thermo-stable PG isoform.
Antisense RNA
It has been found in both procaryotes and eukaryotes, that the production of specific endogenous proteins can be inhibited by use of an antisense RNA. An "antisense RNA" is a complementary version of a naturally occurring or endogenously produced RNA. Because of its complementary sequence, the antisense RNA will hybridize to the mRNA of the protein sought to be inhibited under physiological conditions. This hybridization prevents translation and, therefore, protein production. The duplex RNA complex thus formed is eventually degraded by appropriate cellular mechanisms, without resulting in expression of a protein. An antisense RNA can conveniently be formed for a known protein coding region by reversing the orientation of the protein coding region so that the end that is normally transcribed last is now transcribed first.
Investigators have inhibited production of the catalytic PG2 polypeptide by antisense RNA technology and have shown a greater than 92% reduction of total PG activity relative to wild-type activity levels (Kramer, et al., Horticultural Biotechnology, 1990, pp. 347-355, Wiley-Liss, Inc.). This reduction had significant effects on processed tomato product viscosity when the product was subjected to the normal "hot break methods. These investigators did not, however, determine which PG isoforms were produced in the transgenic fruit. One would expect that the PG2 polypeptide levels were greatly reduced and that all of the PG activity formed would be in the PG1 (heat-stable) isoform due to the presence of existing beta-subunit protein, which would not have been affected by antisense inhibition of PG2 protein.
Inhibiting the level of PG2 expression is not the equivalent of lowered levels of PG1. The reduction of catalytic PG2 polypeptide levels results in the lowering of total PG activity levels and PG2 protein levels, without affecting the formation of the heat stable PG1 isoform directly except by reducing the amount of PG2 available to form PG1. Because 100% inhibition of PG2 production has not been reported, any residual PG2 produced in PG2 antisense plants would associate with the existing beta-subunit protein to produce PG1. Heat inactivation of this remaining PG1 should still require a significant energy input.
What is needed is a transgenic tomato with lowered levels of PG1, the most heat-stable PG isoform.